The field of transgenics was initially developed to understand the action of a single gene in the context of the whole animal and the phenomena of gene activation, expression, and interaction. This technology has also been used to produce models for various diseases in humans and other animals and is amongst the most powerful tools available for the study of genetics, and the understanding of genetic mechanisms and function. From an economic perspective, the use of transgenic technology to convert animals into “protein factories” for the production of specific proteins or other substances of pharmaceutical interest (Gordon et al., 1987, Biotechnology 5: 1183–1187; Wilmut et al., 1990, Theriogenology 33: 113–123) offers significant advantages over more conventional methods of protein production by gene expression.
Heterologous nucleic acids have been engineered so that an expressed protein may be joined to a protein or peptide that will allow secretion of the transgenic expression product into milk or urine, from which the protein may then be recovered. These procedures have had limited success and may require lactating animals, with the attendant costs of maintaining individual animals or herds of large species, including cows, sheep, or goats.
Historically, transgenic animals have been produced almost exclusively by microinjection of the fertilized egg. The pronuclei of fertilized eggs are microinjected in vitro with foreign, i.e., xenogeneic or allogeneic, heterologous DNA or hybrid DNA molecules. The microinjected fertilized eggs are then transferred to the genital tract of a pseudopregnant female (e.g., Krimpenfort et al., in U.S. Pat. No. 5,175,384).
One system that holds potential is the avian reproductive system. The production of an avian egg begins with formation of a large yolk in the ovary of the hen. The unfertilized oocyte or ovum is positioned on top of the yolk sac. After ovulation, the ovum passes into the infundibulum of the oviduct where it is fertilized, if sperm are present, and then moves into the magnum of the oviduct, which is lined with tubular gland cells. These cells secrete the egg-white proteins, including ovalbumin, lysozyme, ovomucoid, conalbumin and ovomucin, into the lumen of the magnum where they are deposited onto the avian embryo and yolk.
The hen oviduct offers outstanding potential as a protein bioreactor because of the high levels of protein production, the promise of proper folding and post-translation modification of the target protein, the ease of product recovery, and the shorter developmental period of chickens compared to other potential animal species. As a result, efforts have been made to create transgenic chickens expressing heterologous proteins in the oviduct by means of microinjection of DNA (PCT Publication WO 97/47739).
The chicken lysozyme gene is highly expressed in the myeloid lineage of hematopoietic cells, and in the tubular glands of the mature hen oviduct (Hauser et al., 1981, Hematol. and Blood Transfusion 26: 175–178; Schutz et al., 1978, Cold Spring Harbor Symp. Quart. Biol. 42: 617–624) and is therefore a suitable candidate for an efficient promoter for heterologous protein production in transgenic animals. The regulatory region of the lysozyme locus extends over at least 12 kb of DNA 5′ upstream of the transcription start site, and comprises a number of elements that have been individually isolated and characterized. The known elements include three enhancer sequences at about −6.1 kb, −3.9 kb, and −2.7 kb (Grewal et al., 1992, Mol. Cell Biol. 12: 2339–2350; Banifer et al., 1996, J. Mol. Med. 74: 663–671), a hormone responsive element (Hecht et al., 1988, E.M.B.O. J. 7: 2063–2073), a silencer element and a complex proximal promoter. The constituent elements of the lysozyme gene expression control region are identifiable as DNAase 1 hypersensitive chromatin sites (DHS). They may be differentially exposed to nuclease digestion depending upon the differentiation stage of the cell. For example, in the multipotent progenitor stage of myelomoncytic cell development, or in erythroblasts, the silencer element is a DHS. At the myeloblast stage, a transcription enchancer located −6.1 kb upstream from the gene transcription start site is a DHS, while at the later monocytic stage another enhancer, at −2.7 kb becomes DNAase sensitive (Huber et al., 1995, DNA and Cell Biol. 14: 397–402).
Scattered throughout the chicken genome, including the chicken lysozyme locus, are short stretches of nucleic acid that resemble features of Long Terminal Repeats (LTRs) of retrovirus. The function of these elements is unclear but most likely help define the DHS regions of a gene locus (Stein et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 6485–6489).
Flanking the lysozyme gene, including the regulatory region, are matrix attachment regions (5′ MAR & 3′ MAR), alternatively referred to as “scaffold attachment regions” or SARs. The outer boundaries of the chicken lysozyme locus have been defined by the MARs (Phi-Van et al., 1988, E.M.B.O. J. 7: 655–664; Phi-Van, L. and Stratling, W. H., 1996, Biochem. 35: 10735–10742). Deletion of a 1.32 kb or a 1.45 kb halves region, each comprising half of a 5 MAR, reduces positional variation in the level of transgene expression (Phi-Van and Stratling, supra).
The 5′ matrix-associated region (5′ MAR), located about −11.7 kb upstream of the chicken lysozyme transcription start site, can increase the level of gene expression by limiting the positional effects exerted against a transgene (Phi-Van et al., 1988, supra). At least one other MAR is located 3′ downstream of the protein encoding region. Although MAR nucleic acid sequences are conserved, little cross-hybridization is seen, indicating significant overall sequence variation. However, MARs of different species can interact with the nucleomatrices of heterologous species, to the extent that the chicken lysozyme MAR can associate with the plant tobacco nucleomatrix as well as that of the chicken oviduct cells (Mlynarona et al., 1994, Cell 6: 417–426; von Kries et al., 1990, Nucleic Acids Res. 18: 3881–3885).
Gene expression must be considered not only from the perspective of cis-regulatory elements associated with a gene, and their interactions with trans-acting elements, but also with regard to the genetic environment in which they are located. Chromosomal positioning effects (CPEs), therefore, are the variations in levels of transgene expression associated with different locations of the transgene within the recipient genome. An important factor governing CPE upon the level of transgene expression is the chromatin structure around a transgene, and how it cooperates with the cis-regulatory elements. The cis-elements of the lysozyme locus are confined within a single chromatin domain (Banifer et al., 1996, supra; Sippel et al., pgs. 133–147 in Eckstein F. & Lilley D. M. J. (eds), “Nucleic Acids and Molecular Biology”, Vol. 3, 1989, Springer.
Deletion of a cis-regulatory element from a transgenic lysozyme locus is sufficient to reduce or eliminate positional independence of the level of gene expression (Banifer et al., 1996, supra). There is also evidence indicating that positional independence conferred on a transgene requires the cotransfer of many kilobases of DNA other than just the protein encoding region and the immediate cis-regulatory elements.
The lysozyme promoter region of chicken is active when transfected into mouse fibroblast cells and linked to a reporter gene such as the bacterial chloramphenicol acetyltransferase (CAT) gene. The promoter element is also effective when transiently transfected into chicken promacrophage cells. In each case, however, the presence of a 5′ MAR element increased positional independency of the level of transcription (Stief et al., 1989, Nature 341: 343–345; Sippel et al., pgs. 257–265 in Houdeline L. M. (ed), “Transgenic Animals: Generation and Use”).
The ability to direct the insertion of a transgene into a site in the genome of an animal where the positional effect is limited offers predictability of results during the development of a desired transgenic animal, and increased yields of the expressed product. Sippel and Steif disclose, in U.S. Pat. No. 5,731,178, methods to increase the expression of genes introduced into eukaryotic cells by flanking a transcription unit with scaffold attachment elements, in particular the 5′ MAR isolated from the chicken lysozyme gene. The transcription unit disclosed by Sippel and Steif was an artificial construct that combined only the −6.1 kb enhancer element and the proximal promoter element (base position −579 to +15) from the lysozyme gene. Other promoter associated elements were not included. However, although individual cis-regulatory elements have been isolated and sequenced, together with short regions flanking DNA, the entire nucleic acid sequence comprising the functional 5′ upstream region of the lysozyme gene has not been determined in its entirety and therefore not employed as a functional promoter to allow expression of a heterologous transgene.
What is still needed, however, is an efficient transcription promoter that will allow expression of a transgene in avian cells that is not subject to positional variation.
What is also needed is a gene expression promoter cassette that will allow expression of a transgene in the oviduct cells of an avian and efficient gene expression regardless of the chromosomal location of the expression system.